Fetal Pig Research Articles

PSI-21 Limited impact of hypothyroidism on porcine fetal growth during mid to late gestation

Abstract In the fetal pig, both circulating thyroid hormone and growth rate are known to increase throughout gestation; however, the functional association between these coincident factors has not been established. Thus, the objective of this project was to investigate this cause-and-effect relationship by evaluating the impact of induced fetal hypothyroidism on fetal growth during mid to late gestation. To induce fetal hypothyroidism, n = 12 pregnant gilts were orally treated with the goitrogen methimazole (MMI) at a dose of 5 mg/kg/d for 21 d, with n = 3 gilts starting treatment on gestation d 34, 45, 55 and 65. An equivalent group n = 12 of gilts were sham treated for the same periods to produce gestationally age-matched euthyroid control litters (CON). Upon completion of the treatment period, gilts were humanely euthanized, and the resulting populations of n = 174 MMI and n = 166 CON fetuses deeply phenotyped. No meconium-stained fetuses were observed at any time point, and only 5 mummified fetuses were observed (3 CON, 2 MMI), indicating treatment has no effect on fetal viability. Fetal body weight (BW) and the weights of individual organs including thyroid, liver, heart, lung, kidney, spleen, and brain were collected. In addition, fetuses were imaged in both left and right lateral recumbency for subsequent morphometric assessment using a custom analysis pipeline in ImageJ. Image analysis yielded parameters including crown-rump length, girth, brow length, depth at the snout/skull junction, depth at the snout/nose junction, and area under the skull curvature (AUC). Left and right measurements for all morphometric parameters were found to be highly correlated (r2 > 0.9) indicating a repeatable measure using this approach. Statistical analysis of all phenotypic differences was carried out using a linear mixed effect model including gestational age and treatment as fixed effects and gilt as a random effect. A significant increase in both absolute and relative thyroid weights indicated the development of goiter and thus, successful induction of hypothyroidism within the MMI litters. In addition, a significant treatment by time interaction was observed, with 0.014 g and 0.21 g increase at d 55 and 66, respectively, indicating reduced compensatory action within the fetal hypothalamic-pituitary-thyroid axis during this earliest period. Liver weight as a percentage of BW decreased from 6.06% to 2.56% between d 55 and 86 in the CON group but was significantly increased at all time points in response to MMI induced hypothyroidism (P < 0.01). A corresponding increase in brain to liver weight ratio over time was suppressed in MMI fetuses (P < 0.05). While all other phenotypic parameters were significantly altered by gestational age, there was no significant impact of fetal hypothyroidism. Collectively, the results indicate that fetal growth during mid to late gestation is not driven by fetal thyroid hormone.

Supplementation of l-arginine in pregnant gilts affects the protein abundance of DNMT1 in 35-day fetuses

Maternal nutrition is one of the main environmental factors regulating gene expression during fetal development through epigenetic modifications. Some nutrients, such as the amino acid l-arginine, are added to maternal diets to modulate gene expression, improve the reproductive performance of females, and enhance conceptus development. This study investigated the hypothesis that supplementation of pregnant gilts with l-arginine regulates gene expression in conceptuses through epigenetic mechanisms. For this, fetal programming phenotypic markers, the expression of key epigenetic genes, and the abundance of DNA methylation proteins (DNMT3A and DNMT1) were evaluated in 25- and 35-day conceptuses from gilts supplemented (ARG) or not (CON) with 1.0 % l-arginine during early gestation. At 25 days, there were no significant differences in phenotypic markers between CON and ARG embryos (P > 0.05). Similarly, no differences were found between CON and ARG fetuses at 35 days (P > 0.05). Maternal supplementation with l-arginine did not influence the expression of the evaluated key epigenetic genes in pig embryos or fetuses, nor DNMT3A protein abundance (P > 0.05); on the other hand, DNMT1 protein abundance was lower in ARG fetuses (P = 0.002). It is concluded that supplementation of l-arginine in pregnant gilts affects epigenetic mechanisms, such as DNA methylation, in 35-day fetuses through regulation of DNMT1 levels. Further studies using transcriptomic and proteomic analysis could reveal additional epigenetic modifications in embryos and fetuses following maternal supplementation with l-arginine.

Epidemiology and Genetic Characterization of Porcine Parvovirus 7 Recovered from Swine in Hunan, China.

Porcine parvovirus 7 (PPV7) was first discovered in swine in 2016, and PPV7 infection has been detected in aborted pig fetuses and in sows that experienced reproductive failure. The objective of this study was to report the prevalence and genetic characterization of PPV7 in Hunan, China. Seventy of the four hundred and twenty-two (16.6%) serum, semen, and tissue samples collected from pigs were positive for PPV7. One complete PPV7 strain and eighteen complete cap gene sequences were obtained; nucleotide and amino acid identity among the nineteen Cap sequences were 88.1-99.4% and 88.1-100%, respectively. They shared identity with previously discovered sequences ranging from 86.6 to 98.9% and 83.7 to 99.8% at the nucleotide- and amino acid-level, respectively. The phylogenetic tree analysis exhibited that PPV7 strains had two major groups based on the presence or absence of five amino acid (181-185) insertions on the Cap protein. Analysis of the Cap protein demonstrated that PPV7 Cap had significant variability, implying that PPV7 evolved at high substitution rates. Substantial variations of that PPV7 Cap may enable the emergence of newly mutated capsid profiles due to its viral adaptation to host responses. Furthermore, antigenic alteration owing to PPV7 Cap protein amino acid mutations at immune epitopes may enable viruses to escape from the host's immune system. This study determined the prevalence and genetic characteristics of PPV7 circulating in swine in Hunan, China, and provided the impetus and basis to further investigate the pathogenicity and epidemiology of PPV7.

Protection from oxygen-glucose deprivation by neurosteroid treatment in primary neurons and oligodendrocytes.

Preterm birth results in an increased risk of neonatal brain injury and neurobehavioural disorders. Despite the seriousness of these adverse outcomes, there are currently no effective therapies to protect the vulnerable developing brain. We propose that neurosteroid replacement therapy may be a novel approach in reducing detrimental neurological outcomes following preterm birth. The use of guinea pig primary neuronal and oligodendrocyte cultures with relevance to late gestation allows insight into the mechanisms behind the effectiveness of these treatments. Primary neuronal and oligodendrocyte cultures were derived from fetal guinea pig frontal cortex brain tissue at gestational age 62 (GA62). Cell cultures were pre-treated with either etifoxine (5µM) or zuranolone (1µm) for 24h prior to insult. Cells were then exposed to either oxygen-glucose deprivation (OGD; 0% O2 and no glucose DMEM; preterm birth insult) or sham (standard cell culture conditions; 25mM DMEM) for 2h. Lactate dehydrogenase assay (LDH) was performed following OGD as a measure of cytotoxicity. Relative mRNA expression of key neuronal and oligodendrocyte markers, as well as neuronal receptors and transporters, were quantified using high throughput (Fluidigm) RT-PCR. OGD significantly increased cellular cytotoxicity in both neurons and oligodendrocytes. Additionally, key neuronal marker mRNA expression was reduced following OGD, and oligodendrocytes displayed arrested mRNA expression of key markers of lineage progression. Treatment with etifoxine restored a number of parameters back to control levels, whereas treatment with zuranolone provided a robust improvement in all parameters examined. This study has demonstrated the neuroprotective potential of neurosteroid replacement therapy in a model of hypoxia related to preterm birth. Neuroprotection appears to be mediated through glutamate reduction and increased brain derived neurotrophic factor (BDNF). Future work is warranted in examining these treatments in vivo, with the overall aim to suppress preterm associated brain damage and reduce long term outcomes for affected offspring.

Enteral plasma feeding improves gut function and immunity in piglets after birth asphyxia.

Birth asphyxia may negatively affect gut function and immunity in newborns. Conversely, immunomodulatory milk diets may protect the gut and immune system against damage caused by asphyxia. Using caesarean-derived pigs as models, we hypothesised that enteral feeding with plasma improves gut and immune functions in asphyxiated newborns. Near-term pig fetuses (98% gestation,) were delivered by caesarean section after 8 min umbilical cord occlusion, leading to transient birth asphyxia (ASP, n = 75) and compared with non-occluded controls (CON, n = 69). Piglets were further randomised to supplementation with/without porcine plasma (plasma, PLA/vehicle, VEH), into bovine colostrum (first 24 h) or formula (until 72 h). Compared with CON, ASP piglets took longer to achieve stable respiration and showed reduced blood pH, weight gain and survival. Independent of asphyxia, plasma supplementation reduced gut haemorrhagic lesions, permeability and inflammatory cytokines together with improved villous morphology and brush-border enzyme activities. Asphyxia reduced blood cytokine responses to ex vivo bacterial stimulation, whereas plasma supplementation ameliorated this effect. Dietary plasma supplementation improves survival, gut functions and immunity in both normal and asphyxiated newborns. The components in plasma that mediate gut-protective effects in piglets remain to be identified, but may benefit also birth-compromised newborn infants. Complicated deliveries leading to birth asphyxia, may negatively affect gut, liver and immune adaptation in the first days after birth. Using a model of birth asphyxia in caesarean-derived piglets, we show that enteral feeding with maternal plasma exerts gut maturational and immunomodulatory effects in both control and asphyxiated animals in the first days of life. The mechanisms behind the gut-protective effects of plasma are unknown, but plasma components hold potential for new oral therapies for compromised newborn infants as well as piglets.

Maternal Uterine Artery Adenoviral Vascular Endothelial Growth Factor (Ad.VEGF-A165) Gene Therapy Normalises Fetal Brain Growth and Microglial Activation in Nutrient Restricted Pregnant Guinea Pigs.

Fetal growth restriction (FGR) is associated with uteroplacental insufficiency, and neurodevelopmental and structural brain deficits in the infant. It is currently untreatable. We hypothesised that treating the maternal uterine artery with vascular endothelial growth factor adenoviral gene therapy (Ad.VEGF-A165) normalises offspring brain weight and prevents brain injury in a guinea pig model of FGR. Pregnant guinea pigs were fed a restricted diet before and after conception and received Ad.VEGF-A165 (1 × 1010 viral particles, n = 18) or vehicle (n = 18), delivered to the external surface of the uterine arteries, in mid-pregnancy. Pregnant, ad libitum-fed controls received vehicle only (n = 10). Offspring brain weight and histological indices of brain injury were assessed at term and 5-months postnatally. At term, maternal nutrient restriction reduced fetal brain weight and increased microglial ramification in all brain regions but did not alter indices of cell death, astrogliosis or myelination. Ad.VEGF-A165 increased brain weight and reduced microglial ramification in fetuses of nutrient restricted dams. In adult offspring, maternal nutrient restriction did not alter brain weight or markers of brain injury, whilst Ad.VEGF-A165 increased microglial ramification and astrogliosis in the hippocampus and thalamus, respectively. Ad.VEGF-A165 did not affect cell death or myelination in the fetal or offspring brain. Ad.VEGF-A165 normalises brain growth and markers of brain injury in guinea pig fetuses exposed to maternal nutrient restriction and may be a potential intervention to improve childhood neurodevelopmental outcomes in pregnancies complicated by FGR.

Time to First Abrasion: A Comparative Evaluation of Flip Flop Strap Designs on Preserved Fetal Pig Skin and Implications for Dorsal Foot Health

Time to First Abrasion: A Comparative Evaluation of Flip Flop Strap Designs on Preserved Fetal Pig Skin and Implications for Dorsal Foot Health

Echocardiographic assessment of cardiovascular physiology of preterm miniature piglets supported with a pumped artificial placenta system.

We evaluated fetal cardiovascular physiology and mode of cardiac failure in premature miniature piglets on a pumped artificial placenta (AP) circuit. Fetal pigs were cannulated via the umbilical vessels and transitioned to an AP circuit composed of a centrifugal pump and neonatal oxygenator and maintained in a fluid-filled biobag. Echocardiographic studies were conducted to measure ventricular function, umbilical blood flow, and fluid status. In utero scans were used as control data. AP fetuses (n=13; 102±4d gestational age [term 115d]; 616±139g [g]; survival 46.4±46.8h) were tachycardic and hypertensive with initially supraphysiologic circuit flows. Increased myocardial wall thickness was observed. Signs of fetal hydrops were present in all piglets. Global longitudinal strain (GLS) measurements increased in the left ventricle (LV) after transition to the circuit. Right ventricle (RV) and LV strain rate decreased early during AP support compared with in utero measurements but recovered toward the end of the experiment. Fetuses supported for >24h had similar RV GLS to in utero controls and significantly higher GLS compared to piglets surviving only up to 24h. Fetuses on a pump-supported AP circuit experienced an increase in afterload, and redistribution of blood flow between the AP and systemic circulations, associated with elevated end-diastolic filling pressures. This resulted in heart failure and hydrops. These preterm fetuses were unable to tolerate the hemodynamic changes associated with connection to the current AP circuit. To better mimic the physiology of the native placenta and preserve normal fetal cardiovascular physiology, further optimization of the circuit will be required.

Fetal external features and morphology of the umbilical cord of wild boars.

This study aimed to describe the gestational and morphological aspects of the fetuses and their respective umbilical cords from two pregnant wild boars (Sus scrofa). Morphological descriptions were provided for 23 fetuses and the gestational ages were estimated through fetal characteristics and formula application. The specimens were fixed in 10% formalin for subsequent macroscopic and microscopic examination. Histological characterization was performed using haematoxylin-eosin (H&E), Masson's trichrome (MT) and Verhöeff's staining techniques. The wild boar fetuses exhibited an estimated gestational age of 55 days (in the larger uterus) and 45 days (in the smaller uterus). They displayed well-developed features consistent with domestic pig fetuses, except for the presence of five pairs of mammae. Additionally, the umbilical cord consisted of two arteries, one vein, an allantoic duct, and a vitelline duct (the latter two identified only microscopically), located in the juxtafetal, intermediate and juxtaplacental portions. The arteries and veins were comprised of endothelium, smooth muscle and collagen fibres, with no elastic fibres observed in the vessel walls. The allantoic duct was lined with simple cuboidal epithelium, while the vitelline duct featured a simple squamous epithelium. In conclusion, the morphological characteristics observed in the examined structures align with the expected patterns for species of the Suidae family. Furthermore, these findings contribute substantially to the morphological characterization of the wild boar, yielding valuable insights into the fetal morphology and the structure of the umbilical cord.

Fetal Kidney Grafts and Organoids from Microminiature Pigs: Establishing a Protocol for Production and Long-Term Cryopreservation.

Fetal organs and organoids are important tools for studying organ development. Recently, porcine organs have garnered attention as potential organs for xenotransplantation because of their high degree of similarity to human organs. However, to meet the prompt demand for porcine fetal organs by patients and researchers, effective methods for producing, retrieving, and cryopreserving pig fetuses are indispensable. Therefore, in this study, to collect fetuses for kidney extraction, we employed cesarean sections to preserve the survival and fertility of the mother pig and a method for storing fetal kidneys by long-term cryopreservation. Subsequently, we evaluated the utility of these two methods. We confirmed that the kidneys of pig fetuses retrieved by cesarean section that were cryopreserved for an extended period could resume renal growth when grafted into mice and were capable of forming renal organoids. These results demonstrate the usefulness of long-term cryopreserved fetal pig organs and strongly suggest the effectiveness of our comprehensive system of pig fetus retrieval and fetal organ preservation, thereby highlighting its potential as an accelerator of xenotransplantation research and clinical innovation.

Effects of foetal size, sex and developmental stage on adaptive transcriptional responses of skeletal muscle to intrauterine growth restriction in pigs

Intrauterine growth restriction (IUGR) occurs both in humans and domestic species. It has a particularly high incidence in pigs, and is a leading cause of neonatal morbidity and mortality as well as impaired postnatal growth. A key feature of IUGR is impaired muscle development, resulting in decreased meat quality. Understanding the developmental origins of IUGR, particularly at the molecular level, is important for developing effective strategies to mitigate its economic impact on the pig industry and animal welfare. The aim of this study was to characterise transcriptional profiles in the muscle of growth restricted pig foetuses at different gestational days (GD; gestational length ~ 115 days), focusing on selected genes (related to development, tissue injury and metabolism) that were previously identified as dysregulated in muscle of GD90 fetuses. Muscle samples were collected from the lightest foetus (L) and the sex-matched foetus with weight closest to the litter average (AW) from each of 22 Landrace x Large White litters corresponding to GD45 (n = 6), GD60 (n = 8) or GD90 (n = 8), followed by analyses, using RT-PCR and protein immunohistochemistry, of selected gene targets. Expression of the developmental genes, MYOD, RET and ACTN3 were markedly lower, whereas MSTN expression was higher, in the muscle of L relative to AW littermates beginning on GD45. Levels of all tissue injury-associated transcripts analysed (F5, PLG, KNG1, SELL, CCL16) were increased in L muscle on GD60 and, most prominently, on GD90. Among genes involved in metabolic regulation, KLB was expressed at higher levels in L than AW littermates beginning on GD60, whereas both IGFBP1 and AHSG were higher in L littermates on GD90 but only in males. Furthermore, the expression of genes specifically involved in lipid, hexose sugar or iron metabolism increased or, in the case of UCP3, decreased in L littermates on GD60 (UCP3, APOB, ALDOB) or GD90 (PNPLA3, TF), albeit in the case of ALDOB this only involved females. In conclusion, marked dysregulation of genes with critical roles in development in L foetuses can be observed from GD45, whereas for a majority of transcripts associated with tissue injury and metabolism differences between L and AW foetuses were apparent by GD60 or only at GD90, thus identifying different developmental windows for different types of adaptive responses to IUGR in the muscle of porcine foetuses.

Cell-Cycle-Specific Autoencoding Improves Cluster Analysis of Cycling Cardiomyocytes.

Our previous analyses of cardiomyocyte single-nucleus RNA sequencing (snRNAseq) data from the hearts of fetal pigs and pigs that underwent apical resection surgery on postnatal day (P) 1 (ARP1), myocardial infarction (MI) surgery on P28 (MIP28), both ARP1 and MIP28 (ARP1MIP28), or controls (no surgical procedure or CTL) identified 10 cardiomyocyte subpopulations (clusters), one of which appeared to be primed to proliferate in response to MI. However, the clusters composed of primarily proliferating cardiomyocytes still contained noncycling cells, and we were unable to distinguish between cardiomyocytes in different phases of the cell cycle. Here, we improved the precision of our assessments by conducting similar analyses with snRNAseq data for only the 1646 genes included under the Gene Ontology term "cell cycle." Two cardiac snRNAseq datasets, one from mice (GEO dataset number GSE130699) and one from pigs (GEO dataset number GSE185289), were evaluated via our cell-cycle-specific analytical pipeline. Cycling cells were identified via the co-expression of 5 proliferation markers (AURKB, MKI67, INCENP, CDCA8, and BIRC5). The cell-cycle-specific autoencoder (CSA) algorithm identified 7 cardiomyocyte clusters in mouse hearts (mCM1 and mCM3-mCM8), including one prominent cluster of cycling cardiomyocytes in animals that underwent MI or Sham surgery on P1. Five cardiomyocyte clusters (pCM1, pCM3-pCM6) were identified in pig hearts, 2 of which (pCM1 and pCM4) displayed evidence of cell cycle activity; pCM4 was found primarily in hearts from fetal pigs, while pCM1 comprised a small proportion of cardiomyocytes in both fetal hearts and hearts from ARP1MIP28 pigs during the 2 weeks after MI induction, but was nearly undetectable in all other experimental groups and at all other time points. Furthermore, pseudotime trajectory analysis of snRNAseq data from fetal pig cardiomyocytes identified a pathway that began at pCM3, passed through pCM2, and ended at pCM1, whereas pCM3 was enriched for the expression of a cell cycle activator that regulates the G1/S phase transition (cyclin D2), pCM2 was enriched for an S-phase regulator (CCNE2), and pCM1 was enriched for the expression of a gene that regulates the G2M phase transition and mitosis (cyclin B2). We also identified 4 transcription factors (E2F8, FOXM1, GLI3, and RAD51) that were more abundantly expressed in cardiomyocytes from regenerative mouse hearts than from nonregenerative mouse hearts, from the hearts of fetal pigs than from CTL pig hearts, and from ARP1MIP28 pig hearts than from MIP28 pig hearts during the 2 weeks after MI induction. The CSA algorithm improved the precision of our assessments of cell cycle activity in cardiomyocyte subpopulations and enabled us to identify a trajectory across 3 clusters that appeared to track the onset and progression of cell cycle activity in cardiomyocytes from fetal pigs.

Integrating Single-Cell and Spatial Transcriptomics Reveals Heterogeneity of Early Pig Skin Development and a Subpopulation with Hair Placode Formation.

The dermis and epidermis, crucial structural layers of the skin, encompass appendages, hair follicles (HFs), and intricate cellular heterogeneity. However, an integrated spatiotemporal transcriptomic atlas of embryonic skin has not yet been described and would be invaluable for studying skin-related diseases in humans. Here, single-cell and spatial transcriptomic analyses are performed on skin samples of normal and hairless fetal pigs across four developmental periods. The cross-species comparison of skin cells illustrated that the pig epidermis is more representative of the human epidermis than mice epidermis. Moreover, Phenome-wide association study analysis revealed that the conserved genes between pigs and humans are strongly associated with human skin-related diseases. In the epidermis, two lineage differentiation trajectories describe hair follicle (HF) morphogenesis and epidermal development. By comparing normal and hairless fetal pigs, it is found that the hair placode (Pc), the most characteristic initial structure in HFs, arises from progenitor-like OGN+/UCHL1+ cells. These progenitors appear earlier in development than the previously described early Pc cells and exhibit abnormal proliferation and migration during differentiation in hairless pigs. The study provides a valuable resource for in-depth insights into HF development, which may serve as a key reference atlas for studying human skin disease etiology using porcine models.

Quantifying Brain Myelin Water Fraction in a Guinea Pig Model of Spontaneous Intrauterine Growth Restriction.

Intrauterine growth restriction (IUGR) is an obstetrical condition where a fetus has not achieved its genetic potential. A consequence of IUGR is a decrease in brain myelin content. Myelin water imaging (MWI) has been used to assess fetal myelin water fraction (MWF) and might potentially assess myelination changes associated with IUGR. To quantify and compare the MWF of non-IUGR and IUGR fetal guinea pigs (GPs) in late gestation. Prospective animal model. Dunkin-Hartley GP model of spontaneous IUGR (mean ± SD: 60 ± 1.2 days gestation; 19 IUGR, 52 control). Eight spoiled gradient-recalled (SPGR) gradient echo volumes (flip angles [α]: 2°-16°), and two sets of eight balanced steady-state free precession (bSSFP) gradient echo volumes (α: 8° - 64°), at 0° and 180° phase increments, at 3.0 T. MWF maps were generated for each fetal GP brain using multicomponent driven equilibrium single pulse observation of T1 /T2 (mcDESPOT). MWF was quantified in the fetal corpus callosum (CC), fornix (FOR), parasagittal white matter (PSW), and whole fetal brain. Linear regression was performed between five fetal IUGR markers (body volume, body-to-pregnancy volume ratio, brain-to-liver volume ratio, brain-to-placenta volume ratio, and brain-to-body volume ratio) and MWF (coefficient of determination, R2 ). A t-test with a linear mixed model compared the MWF of non-IUGR and IUGR fetal GPs (significance was determined at α < 0.05). The MWF of the control fetuses are (mean ± SD): 0.23 ± 0.02 (CC), 0.31 ± 0.02 (FOR), 0.28 ± 0.02 (PSW), and 0.20 ± 0.01 (whole brain). The MWF of the IUGR fetuses are (mean ± SD): 0.19 ± 0.02 (CC), 0.27 ± 0.01 (FOR), 0.24 ± 0.03 (PSW), and 0.16 ± 0.01 (whole brain). Significant differences in MWF were found between the non-IUGR and IUGR fetuses in every comparison. The mean MWF of IUGR fetal GPs is significantly lower than non-IUGR fetal GPs. 2 TECHNICAL EFFICACY: Stage 1.

40 Two-week agriculture institute prepares Mississippi teachers to improve agricultural literacy in classrooms

Abstract Agriculturally rich communities in Mississippi, particularly those with high minority populations, suffer from significant educational disparities, resulting in a median poverty rate 7.8% higher than the national average. Persistent unequal opportunities within the public school system, and a limitation of youth having access to advanced coursework and technology create challenges in improving the welfare of Mississippi communities. Despite being grossly underpaid, teachers in Mississippi remain passionate about agriculture education, often utilizing their personal finances to provide students with opportunities to succeed. Thus, an immersive learning agriculture science professional development program was created for 5th-12th grade classroom teachers. The objective was to improve the quality of education for students by advancing the ability of teachers to foster understanding and practices in agriculture literacy. Teachers (n = 20; cohort 1, n = 12; cohort 2, n = 8) participated in a 2-wk summer institute. Teachers received continuing education credits for their teaching license, a stipend to alleviate associated costs, and supplies for their classroom. The teachers learned agriculture content (e.g., animal, plant, and soil sciences), engaged in hands-on learning labs (e.g., anatomy and immunology), participated in field excursions (e.g., animal handling), received literary instruction, and discussed teacher leadership. To determine if program outcomes were achieved, participants completed surveys (n = 3), interviews (n = 3), and submitted lesson plans (n = 2). Post-institute survey results of participating teachers indicated they were eager to implement their new knowledge in their classrooms, including but not limited to fetal pig dissections, reproductive tract anatomy, blue’s the clue, DNA extraction, carousel activity, mind mapping, and glow germ activity. Statistical analysis using one-way ANOVA was performed on pre- and post-survey knowledge assessment across both cohorts. Regarding agriculture content, self-assessment knowledge scores of both cohorts increased significantly (P ≤ 0.0001) during the post-survey. Both cohort 1 and 2 teachers responded with a score of 80% or higher to: “On an assessment where I had to, “Explain general livestock reproduction and reproductive management practices”; “Explain how a cow's immune system is similar to humans” and, “Explain why farmers work diligently to prevent disease”. In addition, cohort 2 teachers responded with a score of 80% or higher to: “On an assessment where I had to, “Accurately label the anatomical organs of a fetal pig”; “Correctly explain the basic function of fetal pig organs” and, “Explain pregnancy detection in animal science.” This professional development program successfully reached Mississippi teachers, equipping them with tools to increase agriculture literacy. The potential impact of this program extends to encourage more youth to pursue careers in agriculture, foster a more informed consumer base, and mitigate poverty rates within the state. Funding provided by USDA-NIFA-AFRI Education and Workforce Development proposal number 2021-67037-34210/1025666.

Exploration of Preservation Methods for Utilizing Porcine Fetal-Organ-Derived Cells in Regenerative Medicine Research.

Human pluripotent stem cells have been employed in generating organoids, yet their immaturity compared to fetal organs and the limited induction of all constituent cell types remain challenges. Porcine fetal progenitor cells have emerged as promising candidates for co-culturing with human progenitor cells in regeneration and xenotransplantation research. This study focused on identifying proper preservation methods for porcine fetal kidneys, hearts, and livers, aiming to optimize their potential as cell sources. Extracted from fetal microminiature pigs, these organs were dissociated before and after cryopreservation-thawing, with subsequent cell quality evaluations. Kidney cells, dissociated and aggregated after vitrification in a whole-organ form, were successfully differentiated into glomeruli and tubules in vivo. In contrast, freezing hearts and livers before dissociation yielded suboptimal results. Heart cells, frozen after dissociation, exhibited pulsating heart muscle cells similar to non-frozen hearts. As for liver cells, we developed a direct tissue perfusion technique and successfully obtained highly viable liver parenchymal cells. Freezing dissociated liver cells, although inferior to their non-frozen counterparts, maintained the ability for colony formation. The findings of this study provide valuable insights into suitable preservation methods for porcine fetal cells from kidneys, hearts, and livers, contributing to the advancement of regeneration and xenotransplantation research.

Dual isolation of primary neurons and oligodendrocytes from guinea pig frontal cortex.

Primary cell culture is a technique that is widely used in neuroscience research to investigate mechanisms that underlie pathologies at a cellular level. Typically, mouse or rat tissue is used for this process; however, altricial rodent species have markedly different neurodevelopmental trajectories comparatively to humans. The use of guinea pig brain tissue presents a novel aspect to this routinely used cell culture method whilst also allowing for dual isolation of two major cell types from a physiologically relevant animal model for studying perinatal neurodevelopment. Primary neuronal and oligodendrocyte cell cultures were derived from fetal guinea pig's frontal cortex brain tissue collected at a gestational age of 62 days (GA62), which is a key time in the neuronal and oligodendrocyte development. The major advantage of this protocol is the ability to acquire both neuronal and oligodendrocyte cellular cultures from the frontal cortex of one fetal brain. Briefly, neuronal cells were grown in 12-well plates initially in a 24-h serum-rich medium to enhance neuronal survival before switching to a serum-free media formulation. Oligodendrocytes were first grown in cell culture flasks using a serum-rich medium that enabled the growth of oligodendrocyte progenitor cells (OPCs) on an astrocyte bed. Following confluency, the shake method of differential adhesion and separation was utilized via horizontally shaking the OPCs off the astrocyte bed overnight. Therefore, OPCs were plated in 12-well plates and were initially expanded in media supplemented with growth hormones, before switching to maturation media to progress the lineage to a mature phenotype. Reverse transcription-polymerase chain reaction (RT-PCR) was performed on both cell culture types to analyze key population markers, and the results were further validated using immunocytochemistry. Primary neurons displayed the mRNA expression of multiple neuronal markers, including those specific to GABAergic populations. These cells also positively stained for microtubule-associated protein 2 (MAP2; a dendritic marker specific to neurons) and NeuN (a marker of neuronal cell bodies). Primary oligodendrocytes expressed all investigated markers of the oligodendrocyte lineage, with a majority of the cells displaying an immature oligodendrocyte phenotype. This finding was further confirmed with positive oligodendrocyte transcription factor (OLIG2) staining, which serves as a marker for the overall oligodendrocyte population. This study demonstrates a novel method for isolating both neurons and oligodendrocytes from the guinea pig brain tissue. These isolated cells display key markers and gene expression that will allow for functional experiments to occur and may be particularly useful in studying neurodevelopmental conditions with perinatal origins.

Maturation and development of fetal pig intestinal tissue in immunodeficient mice.

This study aimed to compare the degree of maturation and development of fetal pig segmental intestinal tissue with that of spheroids created by in-vitro reaggregation of dissociated fetal intestinal cells after transplantation into immunodeficient mice. Fetal pig small intestines were transplanted as segmental grafts into the omentum and subrenal capsules of immunodeficient mice or enzymatically treated to generate single cells. Spheroids made by in-vitro reaggregation of these cells were transplanted into the subrenal capsules of immunodeficient mice. The segmental grafts and spheroids were harvested four and eight weeks after transplantation, and the structural maturity and in-vivo development of these specimens were histologically evaluated. The spheroids were engrafted and supplied blood vessels from the host mice, but an intestinal layered structure was not clearly observed, and there was almost no change in size. On the other hand, the segmental grafts formed deep crypts in the mucus membrane, the inner circular layer, and outer longitudinal muscles. The crypts of the transplanted grafts harvested at eight weeks were much deeper, and the smooth muscle layer and the enteric nervous system were more mature than those of grafts harvested at the fourth week, although the intestinal peristaltic wave was not observed. Spheroids created from fetal small intestinal cells could not form layered structures or mature sufficiently. Conversely, segmental tissues structurally matured and developed after in-vivo transplantation and are therefore potential grafts for transplantation.

İSLAM HUKUKU AÇISINDAN HARAM KÖKENLİ KATKILARLA ÜRETİLEN AŞILARIN MÜBAH OLMASINDA İSTIHALE VE İSTİHLAK'IN ROLÜ

The emergence of pandemics presents significant challenges to global health, economies, and societies at large. In such critical circumstances, the development and widespread adoption of effective vaccines are of utmost importance to protect lives, improve population health, and restore social and economic stability. Islamic jurisprudence, with its emphasis on the preservation of life and the wellbeing of individuals, plays a crucial role in influencing the acceptance and utilization of vaccines within societies that predominantly adhere to the Islamic faith. This article explores the role of two significant concepts in Islamic jurisprudence, namely Istihalah and Istihlak, in determining the permissibility (halal) of vaccines derived from sources that are considered haram. Focusing on materials such as animal tissues, human fetal tissues, pig gelatin, and alcohol, which may raise concerns re-garding their compliance with Islamic teachings, this article investigates the processes of transformation and purification involved in the production of such vaccines. This article provides a comprehensive analysis of the permissibility of vac- cines derived from haram sources, drawing upon a wide range of scientific opinions, fatwas (religious decrees), classical and contemporary Islamic legal texts, and scientific research. It highlights that the prevailing view among the fatwa councils and the majority of Islamic scholars is that such vaccines are considered halal (permissible). Extensive research demonstrates that the 57 member countries of the Organization of Islamic Cooperation (OIC) exhibit a strong commitment to combating infectious diseases through the prioritized utilization of vaccines. Again, this article adopts a multidisciplinary approach encompassing scientific knowledge, bioethics, and Islamic jurisprudence to elucidate the alignment of vac cination methods with Islamic principles. The principal aim of this study is to enhance the informed decision-making process for healthcare professionals, policy-makers, and religious leaders by furnishing them with invaluable insights.

Abstract 15500: Identifying Cardiomyocytes at Specific Cell-Cycle Stages by a New Cell-Cycle-Specific snRNAseq Analytic Method

Background: Deciphering the cardiomyocyte cell cycle is a critical question in studying heart development and regeneration. We already collected the single-nuclei RNA sequencing (snRNAseq) data from fetal pig hearts and from pigs that underwent apical resection on postnatal day (P) 1 (AR P1 ) followed by myocardial infarction (MI) on P28 (MI P28 ); in these hearts, robust cardiomyocyte proliferation was confirmed. Research questions: Although our previous snRNAseq analyses found cardiomyocyte clusters that primed to proliferate, each of these clusters only appeared at one specific time point; furthermore, these clusters still contain a significant amount of non-cycling cell; and thus, we were unable to distinguish between cardiomyocytes in different phases of the cell cycle. Approach: In this report, we designed a new Autoencoder that only utilized 1646 cell-cycle-specific genes to cluster cardiomyocytes and applied the Autoencoder on pig heart GEO datasets number GSE185289. Results: Five cardiomyocyte clusters (pCM1+2, pCM3-pCM6) were identified. Five proliferation markers (AURKB, MKI67, INCENP, CDCA8, and BRCA5) highly co-expressed in pCM1+2; also, the top enriched gene ontologies of pCM1+2 were all related to cell-cycle. PCM1+2 percentages were ~6% in fetal hearts and ~2-3% in hearts from ARP1MIP28 pigs during the two weeks after MI induction but were nearly undetectable in all other experimental groups and at all other time points. We also identified four transcription factors (E2F8, FOXM1, GLI3, and RAD51) that explicitly coexpressed within pCM1. Also, a pseudo-time trajectory was constructed in Fetal cardiomyocyte, which began at pCM3 (G1/S phase, CCND2 localization), passed through pCM2 (S phase, CCNE2 localization), and ended at pCM1 (M phase, CCNB2 localization). Conclusions: Applying the more precise cell-cycle specific Autoencoder, we found three clusters that likely represented cardiomyocytes at three different cell-cycle stages.